Bacterial strains, genes and enzymes for control of bacterial diseases by quenching quorum-sensing signals

ABSTRACT

The present invention relates to isolated nucleic acid molecules encoding an autoinducer inactivation protein, wherein the encoded protein comprises an amino acid sequence selected from the group consisting of  104 HXHXDH 109 ˜60aa˜H 169 ˜21aa˜D 191  and  103 HXHXDH 108 ˜72aa˜H 180 ˜21aa˜D 202 , and to expression vectors and transformed plant and animal cells comprising the same. The proteins encoded by these nucleic acid molecules provide to a susceptible plant or animal increased resistance to a disease the virulence of which is regulated by autoinducers. Also provided are methods of increasing disease resistance in susceptible plants and animals.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.10/362,569 filed on 21 Jul. 2003, which in turn is a national stagefiling under 35 U.S.C. § 371 of International Patent Application SerialNo. PCT/SG00/00123 filed 23 Aug. 2000. Each application is incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to genes encoding regulators of bacterialmetabolism, more particularly to genes encoding enzymes that quenchquorum-sensing signals. The present invention further relates to methodsof control of bacterial diseases comprising expression of genes encodingautoinducer inhibitors.

BACKGROUND OF THE INVENTION

N-acyl-homoserine lactones, known as autoinducers (AIs), are widelyconserved signal molecules present in quorum-sensing systems of manyGram-negative bacteria. It has been found that AIs are involved in theregulation of a range of biological functions, including bioluminescencein Vibrio species (Eberhard et al., 1981; Cao and Meighen, 1989), Tiplasmid conjugal transfer in Agrobacterium tumefaciens (Zhang et al.,1993), induction of virulence genes in Erwinia carotovora, Erw.chrysanthemi, Erw. stewartii, Pseudomonas aeruginosa, P. solanacerum,and Xenorhabdus nematophilus (Jones et al., 1993; Passador et al., 1993;Pirhonen et al., 1993; Pearson et al., 1994; Beck von Bodman andFarrand, 1995; Flavier et al., 1998; Costa and Loper, 1997; Nasser etal., 1998;), regulation of antibiotic production in P. aureofaciens andErw. carotovora (Costa and Loper, 1997; Pierson et al., 1994),regulation of swarming motility in Serratia liquifaciens (Eberl et al.,1996), and biofilm formation in P. fluorescens and P. aeruginosa(Allison et al., 1998; Davies et al., 1998). Many more bacterial speciesare known to produce AIs, but the relevant biological functions have notyet been established (Bassler et al., 1997; Dumenyo et al., 1998; Cha etal., 1998). Biofilm formation is of particular significance to bacterialpathogenicity, as it makes bacteria more resistant to antibiotics andhost defense responses, and causes microbial contamination in medicaldevices and in drinking water pipelines.

Different bacterial species may produce different AIs. All AIderivatives share identical homoserine lactone moieties, but differ inthe length and structure of their acyl groups. Although the target genesregulated by AIs are extremely varied, the basic mechanism of AIsbiosynthesis and gene regulation seems to be conserved in differentbacteria. The general feature of gene regulation by AIs is cell densitydependence, also known as quorum sensing. At low cell densities the AIsare at low concentrations, and at high cell densities the AIs canaccumulate to a concentration sufficient for activation of relatedregulatory genes (Fuqua and Winans, 1996). The biological functionsregulated by AIs are of considerable scientific, economic, and medicalimportance. New approaches for up or down regulation of bacterial quorumsensing systems would be of significant value, not only in science, butalso in practical applications.

It has been reported recently that a novel gene encoding autoinducerinactivation (aiiA) has been cloned from the Gram-positive bacteriumBacillus sp. strain 240B1 (Dong et al., 2000). Expression of the aiiA intransformed Erw. carotovora strain SCG1, a pathogen that causes soft rotdisease in many plants, significantly reduces the release of AI,decreases extracellular pectrolytic enzyme activities, and attenuatespathogenicity on potato, eggplant, Chinese cabbage, carrot, celery,cauliflower, and tobacco. The results indicate the promising potentialof using the AI-inactivation approach for prevention of diseases inwhich virulence is regulated by quorum sensing signals.

SUMMARY OF THE INVENTION

Bacterial strains and enzymes capable of efficient inactivation ofN-acyl homoserine lactone autoinducers (AIs) are of considerableinterest for biotechnology applications. With the present invention itis disclosed that all Bacillus thuringiensis strains and their closelyrelated species tested were capable of enzymatic inactivation of AIs.One AI synthesis minus mutant of Agrobacterium tumefaciens strain A6,caused by Tn5 insertion mutagenesis, was also found capable of producingAI inactivation enzyme. The genes encoding for AI inactivation enzymeswere cloned either by a functional cloning approach or by a PCR approachfrom the selected bacterial strains. A peptide sequence comparisonindicates that all of these enzymes belong to the metallohydrolasefamily, with amino acid identity ranging from 35.4%-94.0% to thepreviously reported AiiA enzyme. The B. thuringiensis strainseffectively quench AI activity when co-cultured with AI producingpathogenic bacteria, and provide effective biocontrol of potato soft rotdisease caused by Erwinia carotovora. The data suggest that quenchingbiosignals which regulate virulence is an useful strategy for diseasecontrol, and that B. thuringiensis strains which are known forinsecticidal activity are also promising biocontrol agents forprevention of diseases in which virulence is regulated by AIs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the time course of AI (OOHL) inactivation by the proteinextract of A. tumefaciens strain M103. The total protein of M103 wasextracted by sonication disruption of bacterial cells in 1/15 Mphosphate buffer (pH 8.0). Equal volumes of M103 protein extract (1.46mg/ml) and 5000 nM OOHL were mixed and incubated in a 1.5 ml Eppendorfcentrifuge tube at 28° C. Same protein extract was denatured by boilingfor 5 min and used as a control. The samples were taken after 1, 3, 6 hrafter reaction and the reaction was stopped by boiling for 3 min. Thesamples were analyzed for AI activity.

FIG. 2 shows the cloning of the AI inactivation region from the cosmidclones of mutant M103. Two cosmid clones were contained in cosmid vectorpLAFR3 while the four sub-clones in plasmid vector pBluescript II SK(+).Symbols: +, positive in AI inactivation; −, negative in AI inactivation;E: EcoRI; P: PstI.

FIG. 3 shows (A) The potential ORFs in the 1.5 kb AI inactivation regionpredicted with a sequence analysis program; and (B) Deletion analysis todefine the ORF encoding AI inactivation enzyme (AiiB). PCR amplifiedfragments were cloned into vector pBluescript II SK(+) (pBM clones) orin vector pKK223-3 (pKM clones). The numbers under each clone indicatethe start and stop positions of the PCR fragments corresponding to thenucleotide sequences of the 1.5 kb region. All constructs were confirmedby sequencing analysis. The start codon (GTG) and stop codon (TAA) ofthe aiiB ORF are shown under the clone pKM103-315. Solid arrows indicatethe location and direction of lac and tac promoter in these clones, theORFs were indicated with open arrows. Symbols: +, positive AIinactivation activity; −, negative AI inactivation activity.

FIG. 4 shows (A) the nucleotide sequence (SEQ ID NO 1) and (B) predictedpeptide sequence (SEQ ID NO 11) of the aiiB gene cloned from A.tumefaciens M103. The putative ribosome binding (SD) region and two PstIrestriction enzyme sites are underlined, and the putative transcriptiontermination codon is indicated.

FIG. 5 shows the protein sequence comparison of AiiB (SEQ ID NO 11) andAttM (SEQ ID NO 21), a putative protein encoded by the attM gene in theatt region of A. tumefaciens, but its biological function has not beendemonstrated experimentally (GenBank accession No. U59485). These twoproteins exhibit a high degree of similarity (the center sequencerepresents the consensus sequence, four fragments identical to aminoacids 8-43, 45-158, 160-186 and 188-263 of SEQ ID NO 11), but functionalAiiB protein has an additional 7 amino acids in the N-terminus.

FIG. 6 shows a protein sequence comparison of AiiB (SEQ ID NO 11) andAiiA (SEQ ID NO 22), a putative metallohydrolase which inactivates AIcloned from Bacillus sp. 240B1. The two conserved zinc binding regionsare underlined.

FIG. 7 shows the functional cloning of the aiiC gene. (A) Enzymaticinactivation of AI by the suspension culture of Bt strain Cot1. Equalvolume of cell suspension culture (OD₆₀₀=1.1) and 40 μM OOHL were mixedand incubated at 28° C. (▴). The boiled culture and OOHL at sameconcentrations were used as control (▪). The samples were taken at timesas indicated for AI activity assay. (B) Direct subcloning ofAI-inactivation regions from the cosmid clones pLAFR3-aiiC of B.thuringiensis Cot1. The cosmid clone was digested by EcoRI and subclonedinto the pGEM-7Z vector. The AI inactivation positive clone pGEM7-aiiCwas identified by enzyme activity assay. The pGEM7-aiiC was furthersubcloned in the pBluescript II SK(+) vector after BamHI digestion. TheAI inactivation region of about 1.4 kb in size contained in clonepBS-aiiC was completely sequenced. Restriction enzymes: E: EcoRI; B:BamHI.

FIG. 8 shows (A) the nucleotide sequence (SEQ ID NO 2) and (B) predictedpeptide sequence (SEQ ID NO 12) of the aiiC gene cloned from the Btstrain Cot1. The nucleotide sequence of the aiiC ORF is indicated by theuppercase letters and the untranslated regions are indicated by thelower case letters.

FIG. 9 shows the nucleotide sequences and predicted protein sequences ofthe genes aiiD (SEQ ID NOS 3 & 13), aiiE (SEQ ID NOS 4 & 14), aiiF (SEQID NOS 5 & 15), aiiG (SEQ ID NOS 6 & 16), aiiH (SEQ ID NOS 7 & 17), aiiI(SEQ ID NOS 8 & 18), aiiJ (SEQ ID NOS 9 & 19) and aiiK (SEQ ID NOS 10 &20) from Bt strains B1, B2, B17, B18, B20, B21, B22 and B25,respectively.

FIG. 10 shows a phylogenetic tree analysis and amino acid identity of 11cloned AI inactivation genes. The phylogenetic tree was produced byDNASTAR sequence analysis software (DNASTAR Inc.). The distance is shownbelow the tree. The amino acid identity of each sample to AiiA is shownat the right hand of the graph.

FIG. 11 shows the effect of Bt strains on AI production by Erwiniacarotovora. Erw. carotovora SCG1 was inoculated alone in 15 ml LB medium(♦) or co-inoculated respectively in 1:1 ratio with Bt strains Cot1 (Δ)and B1 (), E. coli DH5α (▴) and B. fusiformis (▪) in the same medium.The inoculum concentration (T₀) was 1×10⁷ CFU/ml (colony forming unitper milliliter) for SCG1 and 1×10⁶ CFU/ml for others. After incubationof 1, 2, 3, 4, 6 and 24 hours at 30° C., the bacterial suspensions weretaken and the supernatants were used to bioassay the AI produced. Thedata were means of four repeats.

FIG. 12 shows the effect of Bt strain Cot1 on control of potato soft rotdisease caused by Erw. carotovora SCG1. Dip: Potato slices were dippedinto suspensions of Bt strain Cot1 (C), E. coli DH5α (D) or B.fusiformis (Bf) at a level of about 5×10⁸ CFU/ml or water (W) for about20 sec and then dried in a sterile air flow for about 20 min. The sliceswhich showed no moisture on the surface were inoculated with 2.5 μl ofbacterial suspension containing SCG1 cells equivalent to 5×10⁵ or 5×10⁴CFU. Mixture: the cell culture of SCG1 (2×10⁸ or 2×10⁷ CFU/ml) was mixedrespectively with equal volumes of Cot1 (C), E. coli DH5α (D), B.fusiformis (Bf) (5×10⁸ CFU/ml) or water (W). Two point five microlitresof the mixture was inoculated onto the top of slices. The final cellnumbers of SCG1 inoculated are 2.5×10⁵ or 2.5×10⁴ CFU, as marked in thesecond line below the graph. After 20 hours incubation at 28° C. themaceration area was measured. The data were the means of 4 or 12 (12 forCot1) repeats.

FIG. 13 shows the influence of Bt strains Cot1 and B1 on the growth ofErwinia carotovora SCG1. Erw. carotovora SCG1 (▪) was inoculated aloneor coinoculated with Bt strain Cot1 (♦) and B1 (▴) respectively in a 1:1ratio in 15-ml LB medium. Each strain was inoculated to a finalconcentration of about 1×10⁷ CFU/ml for SCG1 and 1×10⁶ CFU/ml for theothers in the T₀ medium. After 2, 4, 6 and 24 hours culture at 30° C.,the bacterial suspensions were taken and diluted accordingly forspreading on plates for colony counting. The experiment was repeatedfour times and mean data were presented. Top: SCG1, Cot1, and B1 wereincubated and grown separately; Middle: SCG1 was co-incubated with Cot1;Bottom: SCG1 was co-incubated with B1.

FIG. 14 shows changes in bacterial cell numbers (A) and development ofsoft rot symptom (B) on inoculated potato slices. Potato slices weredipped into Cot1 suspensions (5×10⁸ CFU/ml) (▴) or water (♦) for about20 sec and then dried in a Laminar Flow for about 20 min. The sliceswere then inoculated with 5 μl of Erw. carotovora SCG1 (2×10⁹ CFU/ml).After incubation of 1, 2, 3 and 4 days at 28□C, the inoculated sliceswere cut into small pieces and 10 ml of 0.1 M NaCl solution was addedfor resuspension of bacterial cells. The mixture was shaken for 30 minand the suspension was diluted accordingly and spread on to plates forcolony counting. The colony numbers of SCG1 were shown as log10CFU/slice (▴ SCG1 only; ▪ dipped in Cot1) and the numbers of Cot1 (♦)as log 10CFU/mm². The experiment was repeated four times and mean datawere presented.

DETAILED DESCRIPTION OF THE INVENTION

Ten genes encoding AI inactivation enzymes have been cloned from 9 Grampositive bacterial isolates and one Gram negative bacterium (A.tumefaciens). The genes showed different levels of homologies to theaiiA gene, which encodes a putative metallohydrolase with strong AIinactivation activity (Dong et al., 2000). Similar to AiiA, the zincbinding motif regions are highly conserved in the enzyme proteinsencoded by these newly cloned AI inactivation genes. It is very likelythat these ten enzymes are also members of the metallohydrolase family,and use the same molecular mechanism as the AiiA for inactivation ofN-acyl homoserine lactone autoinducers. The present invention furtherenriches the gene pool of AI inactivation enzymes.

In A. tumefaciens, N-acyl homoserine lactone autoinducers, mainly OOHL,are involved in regulation of Ti plasmid conjugal transfer (Zhang etal., 1993). The production of OOHL in A. tumefaciens is induced by theconjugal opines secreted by crown gall tumours (Zhang and Kerr, 1991).The OOHL in turn induces the expression of tra genes. Tra proteins areresponsible for completing the process of Ti plasmid conjugal transfer.Only a few hours are required from opine induction to completion of Tiplasmid conjugal transfer, so the Ti plasmid conjugal transfer cantherefore be regarded as only a transient event. One embodiment of thepresent invention, the aiiB gene for N-acyl homoserine lactonedegradation, identified in A. tumefaciens, highlights the possibilitythat the bacterium has a sophisticated mechanism for control of AIsignal turn over. It is plausible that AI is degraded in Agrobacteriumafter completion of the Ti plasmid conjugal transfer.

It has been noted that a majority of bacterial isolates capable of AIinactivation are Gram positive, belonging to B. thuringenesis andclosely related species. So far, most of the characterisedquorum-sensing signals in Gram-negative bacteria are N-acyl homoserinelactones (Fuqua et al., 1996), while Gram-positive bacteria produceoligopeptides as quorum-sensing signals (Dunny and Leonard, 1997).

Bacillus thuringiensis (Bt) has been used extensively as a microbialinsecticide during the last 30 years. The microorganism is agram-positive, spore-forming soil bacterium, and produces a crystallineparasporal body consisting of one or more crystal (Cry) proteins duringsporulation, which shows biocidal activity against insect families suchas lepidopteran, dipteran, and colepteran insects at larval stages(Lambert and Peferoen, 1992). Some Bt strains have also been reported tobe active against other insect families, as well as mites, nematodes,flatworms, and protozoa (Feitelson et al., 1992). Different Bt strainsproduce more than 28 different but related groups of insecticidalcrystal proteins (http colon slash slash www dot biols dot susx dot acdot uk slash Home slash Neil_Crickmore slash Bt slash). Different groupsof crystal proteins are usually active against a specific spectrum ofinsects, but do not affect other beneficial insects in agriculture.Currently, Bt-based formulations are the most widely used and mosteffective microbial insecticides in agriculture.

As a valuable biocontrol agent, Bt has several advantages including itsspecificity for target insects, its low development cost, and itsenvironmental compatibility (Lambert and Peferoen, 1992). Bt is commonlyfound in natural soil, and normally multiplies by cell division, butforms spores when nutrients are depleted or when the environment becomesadverse. These spores are highly resistant to stress conditions such asheat and drought, enabling the bacterium to survive periods of stress.This sporulating Gram-positive micro-organism can be formulated readilyinto stable products, such as a dry powder, for insect or diseasebiocontrol. Bt also has been subjected to many safety tests, with noharmful effects for animals or human beings.

Bt has not been exploited for disease control because it usually doesnot produce effective antibiotics against bacteria and fungi. In thepresent invention, it has been found that all tested Bt strains arecapable of inactivating AI, and that Bt strains provide effectivebiocontrol against Erw. carotovora infection, whereas B. fusiformis andE. coli strains which do not have AI inactivation genes were unable toprovide biocontrol against Erw. carotovora. Bt strains did not produceany antibiotics and were not inhibitory to the growth of pathogen. Thedata strongly suggest the important role of AI inactivation genes indisease biocontrol. Because the AI diffuses easily into bacterial cells,Bt, capable of eliminating AI constantly from its surroundings, is apromising biocontrol agent, not only for control of plant soft rotdisease caused by Erw. carotovora, but also for control of otherdiseases in which the virulence genes are regulated by AIs.

Accordingly, an object of the present invention is to provide a methodfor increasing resistance in a plant or animal to a disease in whichvirulence is regulated by AIs [such as the diseases caused byPseudomonas aeruginosa, Erwinia stewartii, Erwinia chrysanthemi,Pseudomonas solanacerum, and Xanthomonas campestris (Passador, et al.,1993; Pirhonen, et al., 1993; Pearson, et al., 1994; Beck von Bodman andFarrand, 1995; Barber, et al., 1997; Clough, et al., 1997; Costa andLoper, 1997; Nasser, et al., 1998), and especially plant soft rotdisease caused by Erw. carotovora] comprising administering to the plantor animal an effective amount of a bacterium that is capable ofproducing an autoinducer inhibitor. In a preferred embodiment of thisaspect of the invention, the bacterium administered is a Bacillus sp.,more preferably a variety of Bacillus thuringiensis, most preferably avariety of B. thuringiensis selected from the group consisting of B1,B2, B17, B18, B20, B21, B22 and B25. In another preferred embodiment ofthis aspect of the invention, the animal to be treated is a human.

It is another object of the present invention to provide isolatednucleic acid molecules encoding autoinducer inactivation proteins. Thesenucleic acid molecules encode autoinducer inactivation proteins thatshare the conserved amino acid motif ¹⁰⁴HXHXDH¹⁰⁹˜59aa˜H¹⁶⁹˜21aa˜D¹⁹¹,or the similar motif ¹⁰³HXHXDH¹⁰⁸˜71aa˜H¹⁸⁰˜21aa˜D²⁰². Preferredembodiments of these nucleic acid molecules encode the proteins of SEQID NOS 11-20, and most preferred embodiments of these nucleic acidmolecules have the sequences of SEQ ID NOS 1-10.

Another object of the present invention is to provide an expressionvector that comprises at least one nucleic acid sequence encoding anautoinducer inactivation protein, wherein the encoded protein comprisesthe conserved amino acid motif ¹⁰⁴HXHXDH¹⁰⁹˜59aa˜H¹⁶⁹˜21aa˜D¹⁹¹, or thesimilar motif ¹⁰³HXHXDH¹⁸⁰˜71aa˜H¹⁸⁰˜21aa˜D²⁰², wherein the expressionvector propogates in a procaryotic or eucaryotic cell. Preferredembodiments of these expression vectors comprise at least one nucleicacid sequence encoding a protein having a sequence selected from thegroup consisting of SEQ ID NOS 11-20, and most preferred embodimentshave the nucleic acid sequences of SEQ ID NOs 1-10.

Yet another object of the present invention is to provide a cell of aprocaryote or eucaryote transformed or transfected with an expressionvector of the present invention.

Yet another object of the present invention is to provide an isolatedprotein which has autoinducer inactivation activity, where the proteincomprises the conserved amino acid sequence¹⁰⁴HXHXDH¹⁰⁹˜59aa˜H¹⁶⁹˜21aa˜D¹⁹¹, or the similar motif¹⁰³HXHXDH¹⁰⁸˜71aa˜H¹⁸⁰˜21aa˜D²⁰². Preferred embodiments of the inventioncomprise proteins having the amino acid sequences of SEQ ID NOS 11-20.

Yet another object of the present invention is to provide a method forincreasing disease resistance in a plant or animal, which methodcomprises introducing into a cell of such plant or animal at least onenucleic acid molecule that encodes an autoinducer inactivation proteinin a manner that allows said cell to express said nucleic acid sequence,wherein said autoinducer inactivation protein comprises the conservedamino acid sequence ¹⁰⁴HXHXDH¹⁰⁹˜59aa˜H¹⁶⁹˜21aa˜D¹⁹¹, or the similarmotif ¹⁰³HXHXDH¹⁰⁸˜71aa˜H¹⁸⁰˜21aa˜D²⁰². Preferred embodiments of thisaspect of the invention comprise introducing at least one nucleic acidmolecule encoding a protein having a sequence selected from the groupconsisting of SEQ ID NOS 11-20, and most preferred embodimentscomprising introducing at least one nucleic acid sequence selected fromthe group consisting of SEQ ID NOS 1-10.

Yet another object of the present invention relates to a method ofpreventing or reducing bacterial damage to a plant or animal, whichmethod comprises administering to a plant or animal in need of suchprevention or reduction an effective amount of at least one autoinducerinactivation protein, wherein said protein comprises the conserved aminoacid sequence ¹⁰⁴HXHXDH¹⁰⁹˜59aa˜H¹⁶⁹˜21aa˜D¹⁹¹, or the similar motif¹⁰³HXHXDH¹⁰⁸˜71aa˜H¹⁸⁰˜21aa˜D²⁰². Preferred embodiments of this aspectof the invention comprise providing at least protein having the aminoacid sequences of SEQ ID NOS 11-20.

Yet another object of the present invention relates to a method ofpreventing or reducing the formation of bacterial biofilms, which methodcomprises exposing biofilm-forming bacteria to at least one autoinducerinhibitor protein, wherein said protein comprises the conserved aminoacid sequence ¹⁰⁴HXHXDH¹⁰⁹˜59aa˜H¹⁶⁹˜21aa˜D¹⁹¹, or the similar motif¹⁰³HXHXDH¹⁰⁸˜71aa˜H¹⁸⁰˜21aa˜D²⁰². Preferred embodiments of this aspectof the invention comprise exposing the biofilm-forming bacteria to atleast protein having the amino acid sequences of SEQ ID NOS 11-20.

It is possible to further enhance the efficiency of Aii-producingbacterial strains by using a genetic approach to modify such strains,for example by introducing genes encoding for additional, or moreactive, autoinducer inhibitors. It also is possible to optimise theenzyme activity of aii genes by an in vitro DNA evolution approach.Increasing the expression of Aii enzymes by coupling the aii gene to astrong promoter or increasing the copy number of the aii gene in Btcells would be another useful way to improve the capacity of Bt strainsto quenching AI signals. It is likely that genetically modified Btstrains which secrete AI inactivation enzyme or contain the enzyme inthe outer membrane of the cell could have better efficiencies inquenching AI signals than their wild type parent strain. This isachievable by fusing an aii gene to a sequence encoding a secretion or amembrane attachment signal peptide.

The sequence may be introduced into plant or animal cells by well-knownmethods. Methods for the transformation or transfection of eukaryoticcells with exogenous nucleic acid sequences include transfection,projectile bombardment, electroporation or infection by Agrobacteriumtumefaciens. These methods are likewise familiar to the person skilledin the area of molecular biology and biotechnology and need not beexplained here in detail.

As pathogenic bacteria cells are confined to the intercellular area ofplant tissues, it is desirable to target the Aii protein into theintercellular spaces. Such may be accomplished by fusing a secretionsignal peptide to the Aii protein (Sato, et al., 1995; Firek, et al.,1993; Conrad and Fiedler, 1998; Borisjuk, et al., 1999). Alternatively,a plant membrane attachment motif can be incorporated into the peptidesequence of Aii for anchoring the Aii enzyme in the outer surface ofplant cell membrane.

The present invention also contemplates usage of a bacterial autoinducerinactivation protein directly to treat or prevent bacterial damage. Forexample, the protein may be applied directly to plants in need of suchtreatment or prevention. In a preferred embodiment, the protein isapplied in the form of a composition which comprises an effective amountof the protein and a suitable carrier. The composition may have a widevariety of forms, including solutions, powders, emulsions, dispersions,pastes, aerosols, etc.

The bacterial autoinducer inactivation protein may also be used to treatbacterial infections in animals, including humans. In that application,an effective amount of the active ingredient is administered to ananimal in need of such treatment.

For therapeutic treatment, the active ingredient may be formulated intoa pharmaceutical composition, which may include, in addition to aneffective amount of the active ingredient, pharmaceutically acceptablecarriers, diluents, buffers, preservatives, surface active agents, andthe like. Compositions may also include one or more other activeingredients if necessary or desirable.

The pharmaceutical compositions of the present invention may beadministered in a number of ways as will be apparent to one of ordinaryskill in the art. Administration may be done topically, orally, byinhalation, or parenterally, for example. Topical formulations mayinclude ointments, lotions, creams, gels, drops, suppositories, sprays,liquids and powders. Oral formulations include powders, granules,suspensions or solution in water or non-aqueous media, capsules ortablets, for example. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be used as needed. Parenteralformulations may include sterile aqueous solutions which may alsocontain buffers, diluents and other suitable additives. The dose regimenwill depend on a number of factors which may readily be determined, suchas severity and responsiveness of the condition to be treated.

Traditionally, microbial biocontrol has depended on production ofantibiotics or antimicrobial compounds (Cronin et al., 1997; Liao andSapers, 1999; Emmert and Handelsman, 1999). The present invention offersan alternative strategy for biocontrol, based on quenching biosignalsthat are essential for virulence.

EXAMPLE 1 Bacterial Strains Capable of Inactivating Autoinducers

To identify the genes responsible for inactivation of autoinducersignals, more than 400 field and plant bacterial isolates and about 100stains of the laboratory bacterial culture collection were screened. Thebacterial strains used to test the ability of inactivating autoinducersignals were isolated from soil and plant suspensions as describedpreviously (Dong et al., 2000), or obtained from Bacillus Genetic StockCentre (BGSC) and the American Type Culture Collection (ATCC). Erwiniacarotovora SCG1 was isolated from Chinese cabbage leaves showing softrot symptoms. It was confirmed by 16S DNA sequence and itscharacteristic production of autoinducer and induction of soft rotdisease in potato and Chinese cabbage. These strains were grown at 28°C. in Luria-Bertani (LB) medium with shaking when necessary.Agrobacterium tumefaciens strains were grown at 28° C. in YEB, in BMminimal medium (basic minimal nutrient added with mannitol as solecarbon source), or on nutrient agar plates (Difco Laboratories).Mannitol at a final concentration of 0.2% was used as the sole carbonsource in the minimal medium. Escherichia coli strains were grown at 37°C. in LB or on LB agar plates. Antibiotics were added at the followingconcentrations, when required: rifampin at 50 μg/ml, streptomycin at 100μg/ml, ampicillin at 100 μg/ml, kanamycin at 50 μg/ml, and tetracyclineat 10 μg/ml. X-gal (5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside)(Promega) was included in media at 50 μg/ml for detection ofβ-galactosidase enzyme activity.

More than 30 strains showed different levels of AI inactivationactivity. To characterise the unknown isolates, the 16S rRNA sequencesof these isolates were analysed by PCR amplification and subsequentsequencing. The sequence search showed the 16S rRNA sequences of thosestrains capable of inactivating AI are highly homologous to that ofBacillus thuringiensis (Bt).

To test whether other Bacillus strains also have the AI-inactivationability, known strains of B. thuringenesis, B. cereus, B. mycoides, andB. sphaericus were selected for bioassay. For determination of the AIinactivation ability of bacterial strains and isolates, the autoinducer,N-β-oxo-hexanoyl-L-homoserine lactone (OHHL), orN-β-oxo-octanoyl-L-homoserine lactone (OOHL) was added to the over-nightbacterial cultures which were diluted to OD₆₀₀=1.1, or to the proteinextracts, at a final concentration of 20 μM, and incubated at 28° C. for30 min. The AI remaining in the supernatant was then determined aspreviously described (Zhang, 1993; Dong et al., 2000).

Table 1 shows the AI inactivation activities of the selected strains andsome newly identified isolates. All the tested bacterial strains, exceptB. sphaericus and B. fusiformis, eliminated AI (at a concentration of 20μM OHHL) with different levels of enzyme activities. These strainsinclude 13 known Bacillus species (strains starting with a “B” in Table1), 1 known Agrobacterium and 9 Bacillus species identified by 16S rDNAsequence analysis. Among them, 12 bacterial strains showed a high levelof AI-inactivation activity (>30 μM/h/OD₆₀₀); 8 showed a medium level ofactivity (25-30 μM/h/OD₆₀₀); and the A. tumefaciens strain M103 showed alow level of activity (4.5 μM/h/OD₆₀₀). Except for A. tumefaciens, allthese AI-inactivation strains are Gram-positive and belong to B.thuringenesis or its close related species.

TABLE 1 Bacterial strains and their AI-inactivation activity Enzymeactivity Strains Source (μM/h/OD₆₀₀) 28-32 Bacillus thuringiensis Thiswork 32.4 ± 1.1 258-3 Bacillus thuringiensis This work 32.5 ± 1.2 69Bacillus thuringiensis This work 30.9 ± 2.3 60-1 Bacillus thuringiensisThis work 28.2 ± 5.1 250 Bacillus thuringiensis This work 23.4 ± 3.9 262Bacillus thuringiensis This work 23.1 ± 1.5 B18 Bacillus thuringiensisThis work 27.4 ± 3.0 B20 Bacillus thuringiensis This work 32.7 ± 2.4 B21Bacillus thuringiensis This work 33.1 ± 0.8 B22 B. thuringiensis ssp.This work 32.8 ± 1.3 kurstaki* B23 B. thuringiensis ssp. BGSC(4Q7) 26.7± 3.5 Israelensis* B1 B. thuringiensis ssp. BGSC (4A3) 32.5 ± 0.3thuringiensis B2 B. thuringiensis ssp. BGSC (4D1) 33.0 ± 0.6 kurstakiB12 B. thuringiensis ssp. BGSC (4J4) 33.5 ± 0.9 Aizawai B17 B.thuringiensis ssp. Mycogen(PSS2A1) 28.8 ± 4.1 Wuhanensis B25 Bacilluscereus This work 33.7 ± 0.8 B14579 Bacillus cereus ATCC (14579) 31.7 ±0.6 B6462 Bacillus mycoides ATCC (6462) 29.8 ± 2.2 240B Bacillus sp.This work 33.0 ± 1.0 Cot Bacillus thuringiensis This work 25.1 ± 2.4M103 Agrobacterium This work 4.5 tumefaciens 269 Bacillus fusiformisThis work 0 B29 Bacillus sphaericus BGSC (12A4) 0 *Plasmid minus **Equalvolume bacterial suspension (diluted to OD₆₀₀ = 1.1 from overnightcultures) and OHHL (40 μM) were incubated at 28° C. for 30 min and thenOHHL remaining in the supernatant was determined as previously described(Zhang, 1993). The enzyme activity is shown as digested μM of OOHL perhour per OD₆₀₀ of bacterial culture. Values represent mean ± standarddeviation of 4 replicates. Strains starting with a “B” prefix are theknown Bacillus species. Other Bacillus strains were identified by 16SrDNA sequence analysis.

The evidence suggests that the AI-inactivation gene is located inchromosomal DNA but not in a plasmid, because Bt ssp. kurstaki strain B2and its plasmid minus derivative strain B22, both showed a similar levelof enzyme activity. The second plasmid minus strain B23, belonging to B.thuringenesis ssp. Israelensids, was also capable of enzymaticinactivation of AI.

To investigate the genetic diversity of genes for AI-inactivation, therepresentative bacterial strains showing high, medium or low levels ofAI-inactivation activity were chosen for further cloning experiments.

EXAMPLE 2 Functional Cloning of the aiiB Gene from Agrobacteriumtumefaciens Strain M103

The suicide plasmid pSUP10 (Simon et al, 1983) in E. coli SM10 was usedto introduce transposon Tn5 insertions into the genome of A. tumefaciensoctopine strain A6 by the protocol described by Garfinkel and Nester(1980), except that the bacterial suspensions were spread onto BMminimal plates containing kanamycin (100 μg/ml). Total DNA of A.tumefaciens mutant strain M103 was partially digested with EcoRI, the20-30 kb fragments were recovered from lower melting point agarose geland purified. The purified fragments were ligated to the dephosphorizedEcoRI site of the cosmid vector pLAFR3 (Staskawicz et al., 1987). Theligation mixture was packaged with GigapackTMIII XL Packaging Extract(Stratagene) and then transfected into E. coli DH5α. About 2000individual colonies grown on the selective medium containingtetracycline were maintained as the genomic library of A. tumefaciensmutant strain M103. The cosmid clones containing Tn5 were selected onthe medium containing kanamycin and were further assayed for AIinactivation activity by using the bioassay method described above.Subcloning into the sequencing vector pGEM-7Zf(+) was carried out byroutine techniques (Sambrook et al., 1989). Sequencing was performed onboth strands by using the ABI Prism dRhodamine Terminator CycleSequencing Ready Reaction Kit (Perkin-Elmer Applied Biosystems).

Agrobacterium tumefaciens strain A6 produces N-acyl homoserine lactoneautoinducers (AI) which are involved in regulation of Ti plasmidconjugal transfer (Zhang and Kerr, 1991). But its derivative M103 causedby Tn5 insertional mutagenesis is capable of inactivation of AI. (Table1 and FIG. 1). It is likely that the gene encoding for AI degradation instrain A6 is regulated by a negative regulator, and the Tn5 insertionresulted in constitutive expression of the gene for AI inactivation.

Based on the assumption that the AI inactivation gene may be locateddownstream of the Tn5 insertion site, the cosmid clones containing Tn5transposon were selected by the kanamycin resistance phenotype. Twocosmid clones resistant to kanamycin and showing AI inactivationactivity were obtained from the cosmid library of M103. Restrictionanalysis and bioassay showed that a 5.2 kb EcoRI fragment conferred theAI inactivation activity. Further subcloning narrowed down the region toa 1.5 kb PstI fragment (FIG. 2). Sequence analysis showed that severalputative open reading frames (ORFs) starting with ATG or UTG were in thefragment. One of the ORFs showed 96.8% identity in nucleotide sequenceand 98% in amino acid sequence to the attM gene (U59485) of A.tumefaciens identified previously. However, AI inactivation activity wasnot detected when expressing the attM in E. coli via an expressionvector pKK223-3. Deletion analysis of the 1.5 kb fragment showed that a792 bp ORF, its start codon a GTG rather than the normal ATG, encodingfor AI inactivation (FIG. 3). The gene was named as aiiB (FIG. 4). Incomparison with the AttM whose biological function has not beenidentified experimentally, the AiiB has 7 extra amino acids at the Nterminus (FIG. 5). AiiB showed 35.4% identity at the amino acid levelcompared to the previously reported AiiA (FIG. 6).

EXAMPLE 3 Functional Cloning of the aiiC Gene from B. thuringinesisStrain Cot1

The suspension culture of strain Cot1 eliminated AI (20 μM) completelyafter 2 hr incubation, but bacterial cells killed by boiling for 5 minfailed to inactivate AI (FIG. 7A), indicating an enzymatic inactivationmechanism. To identify the gene encoding for AI inactivation from Cot1,a cosmid library was constructed by EcoRI partial digestion of thegenomic DNA of the bacterial isolate Cot1. Genomic DNA was extractedfrom bacterial isolate Cot1 and digested partially with EcoRI. The DNAfragments were ligated to the dephosphorylized EcoRI site of cosmidvector pLAFR3. Ligated DNA was packaged and transfected into E. coliDH5α. Cosmid clones with AI inactivation activity were identified byusing the bioassay method described above. Subcloning into thesequencing vector pGEM-7Zf(+) or pBlueScript SK were carried out byroutine techniques.

One clone showing AI inactivating function was identified from the onethousand cosmid clones screened. Restriction analysis showed that thisclone contains an insert of 24 kb. All five fragments generated by EcoRIcomplete digestion were subcloned into pGEM7 vector. The bioassay ofthese subclones showed that one clone, pGEM7-aiiC with an insert of 5kb, conferred the AI inactivation activity. Further subcloningidentified a 1.4 kb BamHI fragment contained in the clone pBS-AiiC whichwas responsible for the AI inactivation function (FIG. 7B). The completesequence of the clone pBS-AiiC showed that there is an ORF of 750 bpnucleotides (from 166 to 918) which encodes a protein of 250 amino acids(FIG. 8). Cloning of this ORF in the E. coli expression vector confirmedthat it encoded a functional AI inactivation enzyme, designated as AiiC.At the peptide sequence level, the AiiC gene showed 91% and 33%identity, to the AiiA and the AiiB respectively. The aiiC gene has nosignificant similarity to other known sequences in the databases byFASTA and BLAST analysis at either nucleotide or peptide levels.

EXAMPLE 4 The Autoinducer Inactivation Genes in Bt Belong to the SameGene Family

Among the tested bacterial isolates with AI inactivation activity, allexcept the A. tumefaciens strain M103, are Gram positive, and belong toB. thuringiensis (Bt) or closely related bacterial species. The aiiA andaiiC genes from the two Bacillus strains showed a high level ofsimilarity. It is very likely that the aiiA and aiiC genes are highlyconserved among B. thuringiensis strains. DNA hybridisation (Southernblot) analysis was performed using an aiiC fragment as a probe. Thegenomic DNA was isolated from 18 selected bacterial strains, B1 (Bt ssp.thuringiensis), B2, B3 and B4 (Bt ssp. kurstaki), B22 (Bt ssp. kurstakiplasmid minus), B12 (Bt ssp. Aizawai), B16 and B17 (Bt ssp. Wuhanensis),B23 (Bt ssp. Israelensis), and other Bt strains B18, B20, B21, 240B1,471W, and Cot1 as well as B25 and B26 (B. cereus), and B29 (B.sphaericus). Genomic DNA (20 μg) digested with EcoRI was separated byelectrophoresis in 0.8% agarose gel and then the DNA was transferredonto Hybond-N+membrane (Amersham Pharmacia, Biotech.) according tomanufacture's instructions. The 1.4 kb BamHI fragment containing theaiiC codon region was labelled with DIG for use as a probe forhybridisation. After hybridisation at 65° C., the membrane was washedtwice in 2×SSC, 0.1% SDS at room temperature for 5 min, followed bywashing twice in 0.1×SSC, 0.1×SDS at 65° C. for 15 minutes. Afterwashing, the membrane was detected with anti-DIG-AP conjugate, theNBT/BCIP solution was used as colour substrate according tomanufacture's protocol (Boehringer Mannheim).

The result showed that one hybridising band was clearly detected fromall tested strains, except for B29 (B. sphaericus). These resultsindicated that there is a single gene, with sequence similar to aiiC,present in all tested B. thuringiensis strains and its closely relatedspecies B. cereus. This is in agreement with the bioassay data (Table1).

EXAMPLE 5 Cloning of Other AI Inactivation Genes from More BacterialIsolates

Since the genes for AI inactivation are highly conserved, a PCR approachwas used for the cloning of other AI inactivation genes from theselected B. thuringiensis isolates. Genomic DNA isolated from thebacterial isolates B1, B2, B17, B18, B20, B21, B22 and B25 was used astemplate. Primers were designed based on the conserved sequences of the5′ and 3′ ends of the aiiA and aiiC gene. Standard PCR conditions wereused to amplify AI-inactivation genes from the selected bacterialisolates. The primer sequences were: C5f: 5′-ATG GGA TCC ATG ACA GTA AAGAAG CTT TAT-3′; C3r: 5′-GTC GAA TTC CTC AAC AAG ATA CTC CTA ATG-3′. ThePCR reactions were performed for 35 cycles of 30 sec at 94° C., 30 secat 55° C. and 1 min at 72° C. using a Perkin Elmer GenAmp PCR System2400. Two separate PCR reactions were performed to make sure there wasno error in the amplified sequences. The PCR products were purified byusing QIAquick PCR Purfication Kit (QIAGEN) and the purified PCRfragment was ligated to pGEM-T vector (Promega). Clones havinginactivating autoinducer activity were chosen for further study. Twosuch clones from each strain were sequenced. Nucleic acid sequence dataand deduced amino acid sequences were analysed with the DNASTAR™sequence analysis software package (DNASTAR Inc.) and GCG sequenceanalysis software (Genetics Computer Group, Wisconsin). Databasesearches were performed using the BLASTA search algorithm.

FIG. 9 shows the nucleotide and deduced peptide sequences of 8 AIinactivation genes (named aiiD to aiiK) cloned from Bt strains B1, B2,B17, B18, B20, B21, B22 and B25 respectively. These sequences allcontain an ORF of 750 bp, which encodes a protein of 250 amino acids.

EXAMPLE 6 The Autoinducer Inactivation Genes are Highly Conserved AmongMembers of Bt and Closely Related Bacillus spp

Except for the aiiB gene, all other genes were cloned from the Grampositive bacterial isolates. Sequence analysis indicates that the aiigenes cloned from the Gram positive bacterial isolates are highlyconserved, with high amino acid identities ranging from 90.4% to 94.0%,in comparison to that of AiiA (FIG. 10). The aiiB gene cloned from theGram negative A. tumefaciens showed less similarity to other aii genesand clustered as a single group in the phytogenetic tree (FIG. 10).These results indicate that the autoinducer inactivation genes arehighly conserved among members of Bt and closely related Bacillus.

In these Aii protein sequences, all except AiiB contain severalinvariant histidines with glutamate residues showing a pattern of¹⁰⁴HXHXDH¹⁰⁹˜59aa˜H¹⁶⁹˜21aa˜D¹⁹¹; the AiiB of A. tumefaciens containsthe similar, but distinct motif ¹⁰³HXHXDH¹⁰⁸˜71aa˜H¹⁸⁰˜21aa˜D²⁰².

This pattern agrees with the metallohydrolase criterion (Vallee andGaldes, 1984). The motif HXHXDH in the Arabidopsis glyoxalase II wassuggested to be involved in binding to zinc ion (Crowder et al., 1997).Site-directed mutagenesis has shown that all these residues except thefirst histidine (¹⁰⁴H in AiiA) in this motif are necessary for AiiAactivity. These invariant histidines and glutamate residues are alsopresent in AiiB to AiiK, indicating they belong to the same group ofautoinducer metallohydrolases.

EXAMPLE 7 Effect of Bt Strains on AI Production by Erwinia carotovora

To test the effect of Bt strains on quenching AI production bypathogenic bacteria, Erw. carotovora SCG1 was co-cultured with Btstrains Cot1, B1, E. coli DH5α, and B. fusiformis respectively. AI wasassayed as in Example 1. The AI produced by strain SCG1 was detectedafter 2 hours incubation, and a rapid increase was observed from 2 to 6hours incubation (for cell numbers, see FIG. 14), whereas no AI wasdetected in the culture supernatant of SCG1 co-cultured with eitherCot1or B1 strain, which produce AI inactivation enzymes. In theco-culture supernatants of SCG1 with either E. coli DH5α or B.fusiformis, which do not contain aii genes, AI production levels weredetected that were similar to those observed with SCG1 culture alone(FIG. 11). These results indicate that Bt strains effectively quench AIsignals produced by the pathogen Erw. carotovora SCG1 when the two arecultured together.

EXAMPLE 8 Effect of Bt Strains on the Pathogenesis of Erwinia carotovora

It is known that AI play a key role in regulation of the virulencedeterminates of several pathogenic bacterial species. Since Bt strainseffectively quenched AI signals produced by the pathogen, it is likelythis new function of Bt strains can be exploited for disease control. Totest this possibility, the effect of Bt strains for biocontrol againstplant soft rot disease was investigated. Potato (Solanum tuberosum L.cv. Bintje) tubers were obtained from local stores. After rinsing in tapwater and drying on paper towel, potato tubers were surface-sterilizedwith 70% ethanol, and then were sliced evenly to a 3 mm thickness. Forthe dip treatment, the potato slices were dipped into the bacterialsuspension of Cot1, or other bacterial strains, diluted to aconcentration of 5×10⁸ colony forming unit (CFU) per ml, for about 20seconds. Sterilised water was used as a control. The slices were driedin a laminar flow cabinet for about 20 min to remove surface moisturebefore inoculation with 2.5 μl of Erw. carotovora SCG1 bacterialsuspension containing approximately 2×10⁸ or 2×10⁷, CFU/ml onto the topof each slice. For the mixture treatment, equal volumes of each testingorganism (5×10⁸ CFU/ml), or sterile water were mixed with Erw.carotovora SCG1 bacterial suspension (2×10⁸ or 2×10⁷ CFU/ml). Themixture (2.5 μl) was inoculated to a cut surface of the potato slices.All the potato slices were incubated in a Petri dish at 28° C.Maceration area was measured during incubation. Each treatment wasrepeated 4 to 12 time (12 for Cot1), each repeat was inoculated 3 placeson one slice. For the colonisation experiment, each treatment wasrepeated 4 times, each tuber slice was inoculated only once at thecentre of slice. Potato tuber slices were either treated with Bt strainCot1or other controls first before inoculation of Erw. carotovora SCG1,or SCG1 bacteria were mixed with Cot1or other controls beforeinoculation onto potato slices.

Erw. carotovora SCG1 caused severe tissue maceration of potato slices 20hr after inoculation, whereas on Bt strain Cot1 pre-treated potatoslices the maceration symptom was significantly attenuated (FIG. 12).Co-inoculation of SCG1 with the Bt strain Cot1 also attenuated soft rotsymptoms, especially at the lower concentration of inoculum. Incontrast, control treatments, either pretreatment of potato slices withE. coli or B. fusiformis before inoculation of SCG1, or co-inoculationof SCG1 with E. coli and B. fusiformis respectively, showed severetissue maceration symptoms (FIG. 12). These results suggest that Btstrains could be used as biocontrol agents against soft rot disease inplants.

EXAMPLE 9 In Vitro Competition Between Bt Strain and Erwinia carotovoraSCG1

The Bt strains Cot1 and B1 were tested for production antibioticsagainst Erw. carotovora SCG1. Competition experiments were conducted byco-inoculation of the Bt strain and Erw. carotovora in a 1:1 ratio. Eachstrain was inoculated at the level of about 1×10⁷ CFU/ml for Erw.carotovora and 1×10⁶ CFU/ml for other strains. The mixture was incubatedat 30° C. At different time points the bacteria samples were taken forbioassay of AI production (the bioassay performed as in Example 1), andwere diluted in suitable concentrations to spread on plates for colonycounting. The experiment was repeated four times. For the colonisationexperiment, the potato slices inoculated with Erw. carotovora were takenat times as indicated, and plant tissues about 15×15 mm circling theinoculation site were cut. The cut tissues were cut into small piece andplaced in 10 ml of 01M NaCl. After shaking for 30 min, the supernatantwas diluted in suitable concentrations. Viable numbers of bacterialcells were counted.

On plates of both rich and minimum media, Bt strains did not show anyinhibitory effect on the growth of SCG1. When strain SCG1 and Bt strainCot1or B1 were coinoculated, both Bt strains and SCG1 grew normally,showing the same growth trend over a 24 hr period (FIG. 13).

EXAMPLE 10 Effect of Bt Strain on Colonisation of Tuber Slice by Erwiniacarotovora

To investigate colonisation of Erw. carotovora SCG1 on potato slicesafter incubation, an expression vector containing the GFP gene wastransformed into strain SCG1. The expression vector can be maintained instrain SCG1 stably without selection pressure. There was no differencein virulence between the SCG1 (GFP) and the wild-type SCG1. Toinvestigate the effect of Bt bacteria on the survival and growth of SCG1on plants, potato tuber slices were either dipped into bacterialsuspensions of Cot1, then inoculated with SCG1(GFP), or simultaneouslyinoculated with SCG1(GFP) and Cot1. Changes in bacterial cell numbersand development of soft rotting symptoms of potato tissue were monitoreddaily for 4 days. Results showed that there were no big changes in cellnumbers between SCG1(GFP) on the Cot1-treated slices and the SCG1(GFP)on the water-treated slices during 4-days incubation (FIG. 14). Theresult indicates that Bt strain Cot1 did not significantly affect thegrowth of SCG1(GFP) on the potato tube slices, suggesting thatattenuation of the virulence of Erwinia SCG1(GFP) by Bt strain Cot1 wasnot due to inhibition of SCG1(GFP) cell growth.

REFERENCES

-   Allison, D., Ruiz, B., SanJose, C., Jaspe, A., and Gilbert, P.    (1998). Extracellular products as mediators of the formation and    detachment of Pseudomonas fluorescens biofilms. FEMS Microbiol Lett    167, 179-184.-   Bassler, B. L., Greenberg, E. P., and Stevens, A. M. (1997).    Cross-species induction of luminescence in the quorum-sensing    bacterium Vibrio harveyi. J Bacteriol 179, 4043-4045.-   Beck von Bodman, S., and Farrand, S. K. (1995). Capsular    polysaccharide biosynthesis and pathogenicity in Erwinia stewartii    require induction by an N-acylhomoserine lactone autoinducer. J    Bacteriol 177, 5000-5008.-   Cao, J. G., and Meighen, E. A. (1989). J. Biol. Chem. 264,    21670-21676.-   Cha, C., Gao, P., Chen, Y. C., Shaw, P. D., and Farrand, S. K.    (1998). Production of acyl-homoserine lactone quorum-sensing signals    by gram-negative plant-associated bacteria. Mol Plant Microbe    Interact 11, 1119-1129.-   Costa, J. M., and Loper, J. E. (1997). EcbI and EcbR: homologs of    LuxI and LuxR affecting antibiotic and exoenzyme production by    Erwinia carotovora subsp. betavasculorum. Can J Microbiol 43,    1164-1171.-   Cronin, D., Moenne-Loccoz, Y., Fenton, A., Dunne, C., Dowling, D.    N., and O'Gara, F. (1997). Ecological interaction of a biocontrol    Pseudomonas fluorescens strain producing 2,4-diacetylphloroglucinol    with the soft rot potato pathogen Erwinia carotovora subsp.    atrosetica. FEMS Microbiol Ecology 23, 95-106.-   Crowder, M. W., Maiti, M. K., Banovic, L., and Makaroff, C. A.    (1997). Glyoxalase II from A. thaliana requires Zn(II) for catalytic    activity. FEBS Lett 418, 351-354.-   Davies, D. G., Parsek, M. R., Pearson, J. P., Iglewski, B. H.,    Costerton, J. W., and Greenberg, E. P. (1998). The involvement of    cell-to-cell signals in the development of a bacterial biofilm.    Science 280, 295-298.-   Dong, Y.-H., Xu, J.-L., Li, X.-C., and Zhang, L.-H. (2000). AiiA, a    novel enzyme inactivates acyl homoserine-lactone quorum-sensing    signal and attenuates the virulence of Erwinia carotovora. Proc.    Natl. Acad. Sci. USA 97: 3526-3531.-   Dumenyo, C. K. M., Chun, A. W., and Chatterjee, A. K. (1998).    Genetic and physiological evidence for the production of N-acyl    homoserine lactones by Pseudomonas syringae pv. syringae and other    fluorescent plant pathogenic Pseudomonas species. Eur J Plant Pathol    104, 569-582.-   Dunphy, G., Miyamoto, C., and Meighen, E. (1997). A homoserine    lactone autoinducer regulates virulence of an insect-pathogenic    bacterium, Xenorhabdus nematophilus (Enterobacteriaceae). J    Bacteriol 179, 5288-5291.-   Eberhard, A., Burlingame, A. L., Eberhard, C., Kenyon, G. L.,    Nealson, K. H., and Oppenheimer, N. J. (1981). Biochemistry 20,    2444-2449.-   Eberl, L., Winson, M. K., Sternberg, C., Stewart, G. S. A. B.,    Christiansen, G., Chhabra, S. R., Bycroft, B., Williams, P., Molin,    S., and Givskov, M. (1996). Involvement of N-acyl-L-homoserine    lactone autoinducers in controlling the multicellular behaviour of    Serratia liquefaciens. Mol Microbiol 20, 127-136.-   Emmert, E. A. B., and Handelsman, J. (1999). Biocontrol of plant    disease: a (Gram-) positive perspective. FEMS Microbiol Lett 171,    1-9.-   Feitelson, J. S., Payne, J., and Kim L. (1992). Bacillus    thuringiensis: insects and beyond. Bio/Technology 10, 271-275.-   Flavier, A. B., Schell, M. A., and Denny, T. P. (1998). An RpoS    (sigmaS) homologue regulates acylhomoserine lactone-dependent    autoinduction in Ralstonia solanacearum. Mol Microbiol 28, 475-86.-   Fuqua, C., and Winans, S. C. (1996). Conserved cis-acting promoter    elements are required for density-dependent transcription of    Agrobacterium tumefaciens conjugal transfer genes. J Bacteriol 178,    435-40.-   Garfinkel, D. J., and Nester, E. W. (1980). Agrobacterium    tumefaciens mutants affected in crown gall tumorigenesis and    octopine catabolism. J Bacteriol 144, 732-43.-   Jones, S. M., Yu, B., Bainton, N. J., Birdsall, M., Bycroft, B. W.,    Chhabra, S. R., Cox, A. J. R., Golby, P., Reeves, P. J., Stephens,    S., Winson, M. K., Salmond, G. P. C., Stewart, G. S. A. B., and    Williams, P. (1993). The Lux autoinducer regulates the production of    exoenzyme virulence determination in Erwinia carotovora and    Pseudomonas aeruginosa. EMBO J 12, 2477-2482.-   Lambert, B., and Peferoen, M. (1992). Insecticidal promise of    Bacillus thuringiensis. Facts and mysteries about a successful    biopesticide. BioScience 42, 112-122.-   Liao, C.-H., and Sapers, G. M. (1999). Influence of soft rot    bacteria on growth of Listeria monocytogenes on potato tuber slices.    J Food Prot 62, 343-348.-   Lin, H. C., Lei, S. P., and Wilcox, G. (1985). An improved DNA    sequencing strategy. Anal Biochem 1985 May 15; 147(1):114-9 147,    114-119.-   Nasser, W., Bouillant, M. L., Salmond, G., and Reverchon, S. (1998).    Characterization of the Erwinia chrysanthemi expl-expR locus    directing the synthesis of two N-acyl-homoserine lactone signal    molecules. Mol Microbiol 29, 1391-1405.-   Passador, L., Cook, J. M., Gambello, M. J., Rust, L., and    Iglewski, B. H. (1993). Expression of Pseudomonas aeruginosa    virulence genes requires cell-to-cell communication. Science 260,    1127-1130.-   Pearson, J. P., Gray, K. M., Passador, L., Tucker, K. D., Eberhard,    A., Iglewski, B. H., and Greenberg, E. P. (1994). Structure of the    autoinducer required for expression of Pseudomonas aeruginosa    virulence genes. Proc Natl Acad Sci USA 91, 197-201.-   Piper, K. R., Beck von Bodman, S., and Farrand, S. K. (1993).    Conjugation factor of Agrobacterium tumefaciens regulates Ti plasmid    transfer by autoinduction. Nature 362, 448-450.-   Pirhonen, M., Flego, D., Heikinheimo, R., and Palva, E. (1993). A    small diffusible signal molecule is responsible for the global    control of virulence and exoenzyme production in the plant pathogen    Erwinia carotovora. EMBO J 12, 2467-2476.-   Sambrook, J. F., Fritsch, E. F., and Maniatis, T. (1989). Molecular    Cloning: A laboratory manual (New York: Cold Spring Harbor    Laboratory Press).-   Simon, R., Priefer, U., and Pühler, A. (1983). A broad host range    mobilization system for in vivo genetic engineering: transposon    mutagenesis in Gram-negative bacteria. Bio/Technol. November,    784-791.-   Staskawicz, B. D., Keen, N. T., and Napoli, C. (1987). Molecular    characterization of cloned avirulence genes from race 0 and race 1    of Pseudomonas syringae pv. glycinea. J Bacteriol 169, 5789-5794.-   Vallee, B. L., and Galdes, A. (1984). The metallobiochemistry of    zinc enzymes. Adv Enzymol Relat Areas Mol Biol 56, 283-430.-   Zhang, L.-H. (1993). Molecular biology and biochemistry of a novel    conjugation factor in Agrobacterium. Doctoral Dissertation, The    Adelaide University, Australia.-   Zhang, L.-H., Xu, J., and Birch, R. G. (1998). High affinity binding    of albicidin phytotoxins by the AlbA protein from Klebsiella    oxytoca. Microbiol 144, 555-559.-   Zhang, L.-H., and Kerr, A. (1991). A diffusible compound can enhance    conjugal transfer of the Ti plasmid in Agrobacterium tumefaciens. J    Bacteriol 173, 1867-1872.-   Zhang, L.-H., Murphy, P. J., Kerr, A., and Tate, M. E. (1993).    Agrobacterium conjugation and gene regulation by N-acyl-L-homoserine    lactones. Nature (London) 362, 446-447.

1. An isolated nucleic acid molecule encoding a protein comprising 250 amino acids having the formula 103aa-amino acid motif-59aa, wherein the amino acid motif consists of HXHXDH˜59aa˜H˜21aa˜D, wherein said protein has autoinducer inactivation activity and wherein the protein has an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs:12-20.
 2. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule has a nucleotide sequence selected from the group of nucleotide sequences set forth in SEQ ID NOs:2-10.
 3. An expression vector comprising the nucleic acid molecule of claim
 1. 4. An expression vector comprising the nucleic acid molecule of claim
 2. 5. An isolated protein comprising 250 amino acids having the formula 103aa-amino acid motif-59aa, wherein the amino acid motif consists of HXHXDH˜59aa˜H˜21aa˜D, wherein said protein has autoinducer inactivation activity and wherein the protein has an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs:12-20.
 6. A method of reducing bacterial damage to a plant or animal, which method comprises administering to a plant or animal in need of such reduction an effective amount of the protein of claim
 5. 7. A method according to claim 6, wherein administration is to an animal.
 8. A method according to claim 7, wherein the animal is a human.
 9. An isolated cell of a prokaryote or eukaryote stably transformed with a nucleic acid molecule encoding a protein comprising 50 amino acids having the formula 103aa-amino acid motif-59aa, wherein the amino acid motif consists of HXHXDH˜59aa˜H˜21aa˜D, wherein said protein has autoinducer inactivation and wherein the protein has an amino acid sequence selected from the group consisting of the amino acid sequences set forth in SEQ ID NOs:12-20.
 10. The isolated cell of claim 9, wherein the nucleic acid molecule has a nucleotide sequence selected from the group of nucleotide sequences set forth in SEQ ID NOs:2-10. 